1. Field of the Invention
This invention relates to a method and an apparatus for eluting, by electrophoresis, a selected fraction or fractions present in an electrophoresis carrier or separation gel containing several discrete bands of different fractions.
2. Description of the Prior Art
Excellent fractionations or separations of mixtures of various substances, such as proteins, glycoproteins, nucleotides, nucleic acids, etc., are obtained by electrophoresis using gels as electrophoretic separation matrices. Gels of various materials such as polyacrylamide, starch, agar and gelatin are used for this purpose. Polyacrylamide gels, in the form of elongated cylinders or flat slabs, are particularly preferred for this purpose.
In gel electrophoresis, particles having an effective electrical charge migrate in the gel by the action of an electrical field (potential gradient) applied across the gel. Particles of different charge migrate at different speeds through the gel. Further, referring to particles of equal charge, during their migration through the pores of the three dimensional gel structure, the larger molecules move more slowly than do the smaller molecules because of the molecular sieve effect. Thus the mixture of starting components is fractionated sharply owing to both the differences of the electrical charges of the components of the starting mixture and the differences in their molecular sizes. As a consequence of the electrophoresis, the individual components of the starting mixture are present in the gel as separate, longitudinally spaced, generally transversely extending, distinct bands or zones. These zones can have various widths, measured in the longitudinal direction of the gel, in the range of from about 1 mm to about 1.5 cm. These gel-bound separated fractions can be detected or rendered more readily visible by incubating the gel with fixative-stain solutions. Alternatively, the starting mixture is labelled with a radioisotope and the separated fractions are located by detection of the radioisotope. The gels can be subjected to various analytical determinations in accordance with conventional practices.
It is desired to recover separately individual fractions present as bands in the separation gel, so as to obtain the individual fractions in condition suitable for further analysis. Techniques are known for extraction of a selected gel fraction by cutting the gel cylinder or slab into separate segments, homogenizing each segment (or a plurality of corresponding gel segments from several separation gels) and then eluting same with water or buffer solution and separating the eluate by centrifugation or filtration. The yields of this process are low. In another method of elution, each gel segment (or corresponding gel segments from several separation gels) is homogenized and then is mixed with a new gel solution which is polymerized to form a new gel cylinder or slab. The new gel is subject to electrophoresis until the fraction is swept from the gel by free flow electrophoresis. This process is complex, the yield is poor and extensive dilution of the extracted fraction occurs.
A technique is also known for continuous separation and elution by electrophoresis. This technique is useful as a preparative method wherein milligram quantities of the fractions are recovered. The equipment is complicated and expensive. It is not well adapted for use in the laboratory for effecting microanalytical separations (microgram scale) or ultramicroanalytical separations (nanogram or picogram scale).
Accordingly it is an object of this invention to provide an improved method, and an apparatus for practicing the method, for recovering separately and in a high yield the individual fractions of solutes present in an electrophoresis separation gel.
It is a further object of this invention to provide an improved method, as aforesaid, which is especially adapted for effecting microanalytical and ultramicroanalytical separations, and which provides a high recovery of the desired fraction.
It is a further object of this invention to provide an improved apparatus, as aforesaid, which is relatively simple and inexpensive, which is easy to use and which is reliable and durable.